| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1998: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1997: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
[Introduction] Tissue inhibitors of metalloproteinases (TIMPs) are potent, newly recognized cell-growth factors in serum, which role is independent of that of being common inhibitors for matrix metalloproteinases. To explore other possible functions of these molecules, we have examined whether TIMPs in FCS affect osteoclastic bone resorption in vitro..
[Methods] TIMP-1-free, TIMP-2-free, and TIMP-1 and -2-free FCS wereprepared by the passage of FCS through the corresponding anti-TIMP monoclonal antibody (mAb) affinity columns. Osteoclast-containing cell populations were prepared from long bones of 1-day-old rabbits, and were seeded in medium 199 on bovine-bone slices. After the cells had been allowed to attach for 1.5 h, the bone slices were cultured 37C for 48 h in experimental media indicated. Resorption lacunae were immunostained with anti-type I collagen antibody, and the total number and area of resorption lacunae were then quantified. In addition, the expression of TIMP-1 and TlMP
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-2 on osteoclasts in boe marrow and odontoclast was studied immunocytochemically by using monoclonal antibody.
[Results and Discussion] At first we looked at the effect of anti-TIMP mAbs on the bone resorption by osteoblasts. When anti-TIMP-lor anti-TLMP-2 mAb was added to the culture medium containing 10% FCS, significant suppression of bone resorption was observed with anti-TIMP-2 mAb but not with anti-TIMP-1 mAb. Then we checked the effect of TIMP-free FCSs on the bone resorption. The bone resorption was significantly suppressed in either TIMP-2-free FCS or TIMP-1 and -2-free FCS but little in TIMP-1-free FCS suggesting that TIMP-2 is more effective than TIMP-1. These results are consistent with those obtained by the addition of anti-TIMP mAbs. The activity was almost fully restored by the addition of human recombinant TIMP-1 or/and TIMP-2. The immunocytochemical study reveiled that TIMP-1 and TIMP-2 localized in the cytoplasm in osteoclasts and odontoclasts.To clarify the mechanism of TIMP-dependent bone resorption, we counted and compared the number of trap-positive and multinuclear cells in each culture medium c otaining either 10% FCS or TIMP-1-free and/or TIMP-2-free FCS.There were essentially no difference in the number among them suggesting possible TIMPs role in the functional expression of matured osteoclasts. Less
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